فصلنامه زیست شناسی میکروارگانیسم ها
پیاپی 10 (تابستان 1393)

In this study, Mutation and Selection technique was used to increase production of Rennet (milk clotting enzyme) in Rhizomucor miehei. Mutation and Selection is a technique for microbial strain improvement in which, mutation is induced on an organism by mutagen factors, then superior mutants are selected by screening procedures. Mutations were induced by an optimized dose of UV (as physical mutagen) and Nitrous acid (as chemical mutagen). Superior mutants were selected and compared to the parent strain regarding the clotting activity, proteolytic activity, enzyme production kinetic, yield, productivity and special growth rate in 5 replications. The milk clotting activity of parent strain was 1183 SU.mL-1. Mutants designated U-1113 and N-0227 were isolated which, produced 1771 and 1864 respectively. ratio was measured 4950, 8114 and 8193 SU.PU-1 in parent, U-1113 and N-0227, respectively. Discussion and Production of enzymes in mutants U-1113 and N-0227 were increased 49.7 and 57.6% respectively, relative to parent strain. Yield of production, productivity and C/P ratio were also increased significantly in mutants, regarding the same figures obtained for parent (p<0.05) that shows mutagens were used, have effected on quantity and quality of strain rennet.

“Bioremediation” is one of the most effective methods to remove petroleum contaminants. The aim of the present study is to isolate the indigenous bacteria from the waste petroleum in the Masjed Soleiman No. 9 production tank and to examine the effect of their application on the elimination of petroleum heavy chain hydrocarbons and converting them into light compounds. Two percent of petroleum sludge was inoculated to the mineral basal medium and after proliferation of its indigenous bacteria, they were inoculated into the mixture of oil sludge and sand at level of 5%, and the amount of total hydrocarbons and residual oil were measured and compared. The isolates were identified based on biochemical tests and 16S rRNA gene sequencing. Optimization of nitrogen and phosphate sources was done based on growth curves of selected isolates. Gas chromatography was used to determine degradation of sludge hydrocarbons. In this study, 10 bacterial isolates were isolated from petroleum sludge. Measurement of petroleum total hydrocarbons, using Soxhlet-extraction method, showed that two isolates named MIS1 and MIS2 are able to decompose oil sludge hydrocarbons within 7 days, with the yields of 62% and 72%, respectively. Furthermore, the two isolates reach the end of the logarithmic phase at 48 and 120 hrs, respectively. The best source of nitrogen and phosphate for both isolates was ammonium nitrate and potassium di ­hydrogen phosphate, respectively. The isolates were identified as Arthrobacter aurescens and Pseudomonas aeruginosa, respectively. In gas chromatography analysis it was revealed that Pseudomonas aeruginosa was more potent in degradation of heavy chain hydrocarbons and their conversion to light chain compounds. Discussion and Resident bacteria are present in the oil sludge and are able to degrade the heavy petroleum compounds and convert them into light compounds. These bacteria are effective in the reduction of environmental pollution and production of low molecular weight hydrocarbons.

Many microorganisms like fungi, bacteria and yeasts, have a natural ability to produce vitamins included vitamin B2 or riboflavin. In this regard, the present study was performed to isolation and screening for riboflavin producing yeasts from various sources of soil, leaf and fruit s. Materials and method s: samples of leaf, soil and fruits were prepared for the presence of yeasts and by its ability to produce riboflavin. After purification and enrichment of samples, in order to assay riboflavin production, spectrometry, thin layer chromatography and high performance liquid chromatography were used. Finally, the best selected isolate was identified using conventional morphological, biochemical and molecular techniques. In this study, 26 yeast strains were isolated from environmental samples, that 6 isolates showed the ability to produce riboflavin. Identification results of the best selected isolate by biochemical and phenotypic characteristics revealed that this isolate is related to Clavispora lusitaniae and considering isolation of it from nectarine, has named it Clavispora lusitaniae strain N3 (Gene accession no: JQ586258). Discussion and Although only one of the six producing strains was studied and identified, observation of ability to produce among 23% of strains showed necessity for further investigation. And according to the result of absence of viewing report about production by investigated strain, it can be said that Iran has potentiality for isolation of yeasts and is capable of producing riboflavin.

Clavulanic acid is a major β-lactam antibiotic which is produced by Streptomyces clavuligerus. Clavulanic acid is used in combination of strong but sensitive to β-lactamase antibiotics. The claR gene has an important role in regulation of clavulanic acid production and is needed for the expression of the genes in final step of clavulanic acid biosynthesis. The recombinant construct pMTclaR which contains claR gene is obtained from Isfahan University and plasmid extraction was done from Streptomyces lividans for next steps. The Streptomyces clavuligerus protoplast was prepared and transformation was done by using polyethylene glycol. Transformation was confirmed by plasmid extraction and PCR. Finally, bioassay method was used to survey the effect of extra copy of claR on clavulanic acid production. The typical chalky white colony of Streptomyces clavuligerus was seen on GYME plates containing thiostrepton antibiotic. Plasmid extraction was initially carried out. Furthermore, PCR reaction was done by claR specific primers and the 1334 bp band which was belonging to claR was detected. Finally, the bioassay was done and the diameters of zone of inhibition in control and sample were compared. The results of the bioassay show that amplification of the claR gene in multicopy plasmids resulted in a 2.5 fold increase in clavulanic acid production. Discussion and In this study the 3.3 fold increase in clavulanic acid production was obtained by using an expression vector containing claR. According to the clinical use of clavulanic acid, production of bacterial strains which are able to produce high level of antibiotic can help significantly in customization of antibiotic production.

Trypanosomes infect a variety of animals and cause fever, weakness, and lethargy, which lead to weight loss and anemia. In livestock the disease is fatal unless treated and causes serious economic losses. Present study was conducted to detection of Trypanosoma from one-humped camels slaughtered in Najafabad slaughterhouse. 278 blood samples were taken at three different time periods from one-humped camels slaughtered in Najafabad slaughterhouse and were tested by polymerase chain reaction (PCR) to detection of Trypanosoma. The overall infection rate of Trypanosoma was 1.07%. Positive samples (3.8%) only found in spring and summer, and no positive sample was found in fall and winter. Discussion and Fortunately, this may suggest that the prevalence of Trypanosoma is very low in the region. It is suggested to prevent the disease by preventing livestock suspected of carrying the disease from entering the country. If it is not possible to test imported healthy livestock by molecular assays, it is recommended that simple and rapid tests such as agglutination tests that do not need an expert be assessed.

Pesticides with complex structure have high persistence in ecosystem and biosphere. Pesticides have harmful effects on farmlands, human and natural resources. In this study for isolation of pesticide-degrading bacteria (Fenitrothion) soil samples were collected from pistachio gardens in Kerman province. Collected soil samples were enriched in Bushnell Hass medium with this pesticide as only carbon and energy source. Isolated bacteria were identified by amplification of 16S rDNA gene by PCR and sequencing. In this study three Fenitrothion -degrading bacterial strains were isolated. These isolated bacteria were identified as: Pseudomonas fluorescens strain F1، Bacillus cereus strain F3 and pseudomonas aeruginosa strain F4. The effects of pesticides concentration on each dominant bacterial strain were investigated. For Fenitrothion degrading bacterium (F4 strain) growth continue until 100 ppm and then decreased. The result of Gas Chromatography (GC) analysis confirmed the biodegradation ability of selected bacterial strains. Discussion and The results of this study demonstrated that there is a diversity of pesticide-degrading bacteria (Fenitrothion) in soil ecosystem farmlands of Kerman province. It is seemed by application of these pesticide-degrading bacteria in farmlands and using bioremediation technique the ecosystem contamination of pesticide can be decreased.

Lichens are symbiotic associations of a fungus with green alga or cyanobacteria. Their secondary metabolites are produced by the mycobiont, and accumulate as extracellular tiny crystals on the outer surfaces of the hyphae. Th metabolites have unique biological activities such as antimicrobial, antifungal, antiviral, antiprotozoal, anti-proliferative, anti-tumor, antioxidant, and anti-inflammatory. Protoparmeliopsis muralis was collected from KaneGonbad mountains in Ilam province and identified by using chemical tests and survey of the anatomical characters. Then TLC and HPLC were carried. The excision wound (5cm long and 2mm deep) was created on skin. Then wounds were infected with Staphylococcus aureus by sterile swab. Rats were randomly placed into three groups: 1- control group, 2- treated group with 5 percent ointment of methanolic extract of P. muralis, and 3- treated group with 10 percent ointment of the extract. Finally an amount of wound healing were measured on days 1, 3, 5, 7, 9 and 11. By using Thin layers Chromatography and High Performance Liquid Chromatography, the existence of psoromic acid, usnic acid, zeorin and fumarprotocetraric acids were confirmed. The result of the present study demonstrated that methanolic extract P. muralis considerably decreased the area of wound in the treatment group to the control group. Discussion and Usnic acid is the major compound in the thallus, it seems that this substance is responsible for special anti-microbial effects and wound healing of this species. Methanolic extract of P. muralis accelerates S. aureus infected wound healing process and decreases the necessary duration of complete wound healing.

Cyanide is a toxic and hazardous compound for all organisms which is produced enormously by human being and causes the environment pollution. Biodegradation is the best method for cyanide elimination in industrial wastewater. The aims of this study were isolation of cyanide degrading bacteria from contaminated soil and investigation of their ability for cyanide degradation. After soil samples collection, enrichment of cyanide degrading bacteria was performed in a minimal medium containing 0.5 mM potassium cyanide. The ability of isolated bacterium to utilize the cyanide as sole carbon and nitrogen source was investigated. Cyanide degradation and ammonium production was determined in growth medium using picric acid and Nessler’s regent methods. Toxicity effect of different cyanide compounds on bacterial growth was determined using minimum inhibitory concentration. In addition, the ability of the isolated bacterium to utilize different cyanide compounds was investigated. Identification of the isolate was undertaken using morphological, physiological and biochemical characteristics and molecular analysis. A bacterium with ability to degrade cyanide as sole carbon and nitrogen source was isolated from soil. This bacterium named as isolate MF1. MF1 degraded cyanide in growth medium in alkaline condition after 40 hours. Moreover this isolate tolerated more than 7 mM potassium cyanide. The results showed that there was a direct relation between decreasing of cyanide concentration, increasing of ammonia concentration and growth of MF1. In addition, the isolated bacterium demonstrated the ability to utilize different cyanide compounds as sole carbon and nitrogen source. The results of morphological and physiological characteristics showed that this bacterium belonged to the Serratia sp. Moreover, 16S rDNA sequencing and phylogenetic analyses exhibited that MF1 strain was similar to Serratia nematodiphila with 99% homology. Discussion and The results demonstrated that the isolated bacterium is a suitable candidate for degradation of cyanide in alkaline condition. This bacterium could introduce for treatment of industrial wastewater and sites that contaminated with cyanide.

Many Species of fungi with ability to metabolize of petroleum hydrocarbons are known so far. These fungi are resistant in oil contaminated sites.This investigation aims at studying fungal population diversity in oil contaminated soils of Khuzestan province and identifying fungal flora in these regions. Crude oil contaminated soil samples were collected from different regions of Khuzestan province. For isolation and enumeration of total heterotrophic fungi, Potato Dextrose Agar medium supplemented with streptomycine was used. The isolated fungi were identified via morphological studies, staining by lactophenol cotton blue, observation with a light microscope and comparing with descriptive and canonizative refereces. Total fungal counts ranged from 0.41 × 102 to 3333.33 × 102 CFU/g. Isolated fungi belong to Aspergillus, Penicillium, Fusarium, Candida, Rhodotorula, Aureobasidium, Mucor, Rhizopus and Acremonium. Most dominant genera were Aspergillus and Penicillium. Discussion and Studies on isolation of fungi in oil containing environments showed that, abundance and fungal diversity in different stations significantly were different. The increase in the number of fungi in crude oil soils showes the probability of degradation and consumption of oil contaminated by fungi. Diversity and distribution of soil microbial population are determined by a number of environmental factors such as pH, electrical conductivity and soil organic matter.

Staphylococcus aureus is considered as one of pathogenic agents in humans, that engages different body parts including respiratory system and causes to spend lots of costs and extending patient’s treatment period. This study which is performed to separate and investigate the pattern of antibiotic resistance in Staphylococcus aureus isolates from upper respiratory system infections in Shahrekord. This study was done by sectional-descriptive method On 200 suspicious persons to the upper respiratory system infections who were referred to the Imam Ali clinic in Shahrekord in 2012. After isolation of Staphylococcus aureus from cultured nose discharges, antibiotic resistance genes were identified by polymerase chain reaction (PCR) by using defined primer pairs. Among 200 investigated samples in 60 cases (30%) Staphylococcus aureus infection (by culturing and PCR method) was determined. Isolates showed the lowest amount of antibiotic resistance to vancomycin (0.5%) and the highest amount of resistance to the penicillin G and cefotaxime (100%). mecA gene (encoding methicillin resistance) with frequency of 85.18% and aacA-D gene (encoding resistance to aminoglycosides) with frequency of 28.33% showed the highest and lowest frequency of antibiotic resistance genes coding in Staphylococcus aureus isolates respectively. Discussion and Notable prevalence of resistant Staphylococcus aureus isolates in community acquired respiratory infections, recommend continuous control necessity to impede the spreading of these bacteria and their infections.